Relationship between the Rod complex and peptidoglycan structure in Escherichia coli

Abstract Peptidoglycan for elongation in Escherichia coli is synthesized by the Rod complex, which includes RodZ. Although various mutant strains of the Rod complex have been isolated, the relationship between the activity of the Rod complex and the overall physical and chemical structures of the peptidoglycan have not been reported. We constructed a RodZ mutant, termed RMR, and analyzed the growth rate, morphology, and other characteristics of cells producing the Rod complexes containing RMR. The growth and morphology of RMR cells were abnormal, and we isolated suppressor mutants from RMR cells. Most of the suppressor mutations were found in components of the Rod complex, suggesting that these suppressor mutations increase the integrity and/or the activity of the Rod complex. We purified peptidoglycan from wild‐type, RMR, and suppressor mutant cells and observed their structures in detail. We found that the peptidoglycan purified from RMR cells had many large holes and different compositions of muropeptides from those of WT cells. The Rod complex may be a determinant not only for the whole shape of peptidoglycan but also for its highly dense structure to support the mechanical strength of the cell wall.


| INTRODUCTION
Bacterial cells show a wide variety of cell shapes, such as round, rod, and spiral (Young, 2003;Young, 2010). Each bacterial species has to maintain its shape during various cellular events, including cell division and segregation of genomic DNA. Most bacterial cells are surrounded by peptidoglycan, a macromolecule consisting of glycan strands crosslinked by short peptides. Peptidoglycan determines cell shape because the shape of the purified peptidoglycan is reminiscent of that of the bacterial cells (Egan et al., 2020;Pedro et al., 1997;. Escherichia coli exhibits a rod shape consisting of a central cylinder and polar caps. The synthesis of peptidoglycan is regulated by the Rod complex (Figure 1a), including actin homolog MreB, peptidoglycan synthases penicillin-binding protein (PBP) 2 and RodA, a transmembrane protein RodZ, MreC, and MreD (Ago & Shiomi, 2019;Blaauwen et al., 2008;Egan et al., 2020;. PBP2 is a transpeptidase required for cell elongation (Sauvage et al., 2008;Spratt, 1975) and RodA is a glycosyltransferase (Emami et al., 2017;Meeske et al., 2016;Sjodt et al., 2020). The mreC and mreD genes constitute an operon with the mreB gene, and these gene products are functionally related.
MreC interacts with MreB and MreD, whereas MreD does not interact with MreB (Kruse et al., 2005). MreC also interacts with PBP2 (Contreras-Martel et al., 2017) and this interaction is thought to cause a structural change in PBP2 and stimulate peptidoglycan polymerization and crosslinking (Rohs et al., 2018). It has been shown that the balance between MreC and MreD determines the activity of PBP2 (Liu et al., 2020). RodZ physically and genetically interacts with itself, MreB, MreC, MreD, PBP2, and RodA (Bendezú et al., 2009;Ikebe et al., 2018;Morgenstein et al., 2015;Shiomi et al., 2008. Thus, RodZ interacts with all known major components of the Rod complex and therefore plays a key role in this complex. RodZ forms a "superstructure" of high molecular weight which dissociates into a hexamer, suggesting that the Rod complex consists of several small  RodZ (a, left) or RMR (b, left) and Rod complex without RodZ (ΔrodZ) (c, left). For the structure of each protein, we used the structures registered in the database (https://alphafold.ebi.ac.uk) of proteins predicted by Alpha Fold2 (Jumper et al., 2021). IM, inner membrane. Morphology of cells producing WT RodZ (a, middle and right) or RMR (b, middle and right) and cells lacking rodZ (c, middle and right) and distribution of length and width of each strain (right). Phase contrast images are shown (middle). Blue and magenta lines indicate the average length and width of WT cells, respectively. units including the RodZ hexamer (Mitobe et al., 2020). The Rod complex is highly dynamic; that is, the Rod complex rotates perpendicularly to the long axis of the cell (Domínguez-Escobar et al., 2011;Garner et al., 2011;Teeffelen et al., 2011), allowing the insertion of peptidoglycan in the cell surface layer in an evenly distributed manner. Therefore, if the presence of the Rod complex components and the interactions between the components are not maintained correctly, the peptidoglycan will not be formed correctly, resulting in abnormal morphology. Such morphological abnormalities can cause growth inhibition and cell death.
The transmembrane protein RodZ is not essential for viability but is critical for cell shape maintenance and fast growth in E. coli (Bendezú et al., 2009;Shiomi et al., 2008). Cells lacking rodZ are round or oval in shape and grow slower than WT cells. We previously isolated mutants that suppressed the rodZ phenotypes and found that most of the mutations occurred in mreB, mrdA (encoding PBP2), and mrdB (encoding RodA) . Most of the mutations in mreB are located at the interface between two MreB filaments, so these mutations would strengthen MreB assembly without RodZ, suggesting that RodZ helps in the assembly of MreB filaments in vivo. One of the suppressor mutations (RodA A234T ) found in mrdB was shown to have an increased activity of peptidoglycan synthesis (Rohs et al., 2018). Interestingly, cells producing these suppressor mutants, or PBP2 L61R which suppressed mreC defective mutants, were resistant to A22, which inhibits MreB assembly (Rohs et al., 2018;, suggesting that MreB filament is more stable in these suppressor strains than in WT cells. It is unclear how the effects or signals of mutations in PBP2 or RodA are transmitted to MreB, or what proteins are involved in the process. One of the candidate proteins is RodZ because it interacts with MreB, PBP2, and RodA. In particular, because MreB is a cytoplasmic protein and the active sites of PBP2 and RodA are in the periplasm, the transmembrane domain of RodZ appears to be important for the transmission or connection between MreB and PBP2/RodA through RodZ (Morgenstein et al., 2015).
The chemical structure of peptidoglycan and its synthetic pathways have been studied for many years. The structure of peptidoglycan was visualized using electron microscopy (EM) and atomic force microscopy (AFM). These observations revealed the meshwork structure of peptidoglycan and the arrangement of glycan strands perpendicular to the long axis (de Pedro et al., 1997;Gan et al., 2008;Pasquina-Lemonche et al., 2020;Tulum et al., 2019;Turner et al., 2018). Recently, it was shown by AFM that treatments of E. coli with β-lactam and Staphylococcus aureus with antibiotics such as methicillin and vancomycin result in holes in the peptidoglycan (Elsbroek et al., 2023;Salamaga et al., 2021). If the balance between peptidoglycan synthesis and hydrolysis is not properly maintained, the peptidoglycan structure cannot be maintained and bacterial cells would be lysed. The relationship between the overall structure of the peptidoglycan and the activity of the Rod complex is unclear.
To investigate the relationship between the activity of the Rod complex and the structure of peptidoglycan, we constructed and characterized a chimeric protein of RodZ and MalF, named RMR, in which the transmembrane domain of RodZ was replaced with the corresponding domain of MalF. Cells producing RMR grew slower than WT cells and showed an abnormal shape. The subcellular localization of RMR was different from that of WT RodZ. We isolated suppressor mutations of the slow growth phenotype of RMR, and the suppressors restored rod shape and the localization of the Rod complex containing RMR was rescued to a WT Rod complex localization pattern. Most of the mutations were mapped to components of the Rod complex. We then directly observed peptidoglycan by quick-freeze, deep-etch electron microscopy (QFDE-EM). This method is suitable for observing the bacterial cell surface layer with high resolution (Ojima et al., 2021;Tulum et al., 2019). In particular, the structure of the surface layer (peptidoglycan layer) of Bacillus subtilis and its L-form cells was recently observed with this method (Tulum et al., 2019). Using these methods, we found that peptidoglycan purified from cells producing RMR had more and larger holes than the suppressors. We also analyzed the chemical structures of muropeptide and found that the suppressor mutation certainly restored the chemical structure of muropeptide from RMR-type to WT.

| Strain construction
The primers used for the strain constructions are listed in Table 2. DNA polymerase Phusion or Taq (New England Biolabs) was used for polymerase chain reaction (PCR). Cells producing sfGFP-rmr were constructed as follows: genomic DNA from RU382 (Ikebe et al., 2018) was amplified using primers 1266/841 and 842/18. The second PCR was carried out with these PCR products as templates and primers 1266 and 18. The PCR product was introduced into strain BW25113 carrying pKD46 (Datsenko & Wanner, 2000) by electroporation. Cells were selected on L plates containing 10 µg mL −1 Cm. The resulting strain was transformed with plasmid pCP20 by selection for ampicillin resistance (Amp R ) at 30°C. The strain was then incubated at 42°C in the absence of Amp, and colonies that grew were screened for Amp S and Cm S phenotypes at 37°C. The resulting strain was designated as RU1353. A P1 lysate prepared from DS1317 (Kawazura et al., 2017) was used to transduce mreB-mCherry SW ΔyhdE::cat into RU1353 to yield RU1354 (sfGFP-rmr mreB-mCherry SW ). To transfer suppressor mutations, we performed the procedure as previously described . pKD3 was used as a template and primers 113 and 114 (for yhdE) or primers 287 and 288 (for rlpA) were used for PCR. PCR fragments containing a cat cassette flanked by an FLP recognition target site were inserted between the first and second codons of chromosomal yhdE (for suppressors in mreB, mreC, or mreD) or rlpA (for suppressors in mrdA or mrdB) genes in each suppressor strain carrying the λ Red expression plasmid pKD46 (Datsenko & Wanner, 2000). To transfer mreB, mreC, mreD, mrdA, and mrdB mutations, chloramphenicol-resistant (Cm R ) colonies were isolated after transformation of suppressors, which have mutations in mreB, mreC, mreD, mrdA, or mrdB, with PCR fragments to insert a cat resistance cassette in the yhdE gene. This gene is downstream of mreD (for mreB, mreC, and mreD mutations). A cat resistance cassette was also inserted into the rlpA gene, which is downstream of mrdB (for mrdA and mrdB mutations). This yielded RU1701 (mreB E122D ΔyhdE::cat), RU1647(mreB R124L ΔyhdE::cat), RU1640 (mreB E137G ΔyhdE::cat), RU1641 (mreC S153I ΔyhdE::cat), RU1642 (mreD F123L ΔyhdE::cat), RU1644 (mrdA T52I ΔrlpA::cat), RU1645 (mrdA I59S ΔrlpA::cat), RU1702 (mrdA A201V ΔrlpA::cat), RU1648 (mrdA V227L ΔrlpA::cat), RU1643 (mrdA R234L ΔrlpA::cat), and RU1646 (mrdB K243N ΔrlpA::cat). P1 phage was grown on a donor carrying mreB, mreC, or mreD mutations, and the yhdE gene was inserted with a cat resistance cassette, or mrdA or mrdB mutations, and the rlpA gene was inserted with a cat resistance cassette, and were used to transduce RU383 (sfGFP-RodZ), RU1353 (sfGFP-RMR), or RU2 (ΔrodZ::kan). Fresh transductants were restreaked on L plates containing Cm, and Cm R clones were selected. All the mutation sites were sequenced and confirmed. The resultant strains are listed in Table 1 and were used for further analyses.

| Image analyses
Cells were detected and counted automatically using ImageJ and its plug-in MicrobeJ (Ducret et al., 2016) or EzColocalization (Stauffer et al., 2018). All but overlapping cells in the images were counted.
2.6 | Isolation of suppressors of the slow-growth phenotype of RMR cells Isolation of suppressors of the slow-growth phenotype of RU1353 (sfGFP-RMR) was performed as previously described . Briefly, several different colonies of strain RU1353 were cultured in L medium at 37°C, diluted 100-fold in new L medium the next day, and cultured further at 37°C. After repeating this inoculation for 1 week, the bacterial cells were spread on Lplates. Large and small colonies appeared, and large colonies were isolated as suppressors.

| Whole-genome sequencing and SNP genotyping
Genomic DNA was purified from each suppressor strain using the Wizard Genomic DNA Purification Kit (Promega). One microgram of genomic DNA was sheared using an M220 focused ultrasonicator (Covaris) to obtain peak fragment lengths of 500-600 bp. Next, the  (2000) AGO ET AL.
distilled water and centrifuged at 20,000g for 30 min. Finally, the pellet was suspended in 200 μL of distilled water to obtain a purified PG sample. Purified peptidoglycan was observed using QFDE-EM.
Observations were performed as previously described (Tulum et al., 2019). Pores smaller than 4 nm 2 and larger than 1000 nm 2 were excluded from the quantification analysis.

| Sacculus composition analysis
Peptidoglycan compositions in E. coli strains were analyzed as described previously (Desmarais et al., 2014;Kühner et al., 2014).  We then calculated the growth rate of cells producing sfGFP-RodZ (hereafter simply referred to as RodZ or WT), sfGFP-RMR (hereafter simply referred to as RMR), and cells lacking rodZ as a control. We found that the doubling times of cells producing RodZ and RMR or cells lacking rodZ were 30 min (WT), 36 min (RMR), and 48 min (ΔrodZ) (Table 4).
Then, we examined the shape of cells producing RMR and ΔrodZ cells as a control ( Figure 1 and Table 4). Cells producing RodZ (WT) showed a rod shape, while cells producing RMR or cells lacking rodZ showed a round or oval shape. We measured the cell length and width ( Figure 1a-c, and Table 4). The average length (L) and width (W) ± standard deviation were 2.68 ± 0.72 µm (L) and 1.40 ± 0.24 µm (W) (RMR), and 2.35 ± 0.69 µm (L) and 1.56 ± 0.27 µm (W) (ΔrodZ). As described above, the average cell length (L) and width (W) ± standard deviation of cells producing RodZ were 3.25 ± 0.75 µm (L) and 0.94 ± 0.04 µm (W). These results indicate that RMR did not completely lose the function of RodZ, and was an intermediate phenotype between WT and ΔrodZ.

| Cluster formations of RMR
Next, we observed the subcellular localization of RodZ and RMR using epifluorescence microscopy. To observe sfGFP-RodZ and sfGFP-RMR, which are transmembrane proteins, we attempted to image fluorescence at the cell surface. Therefore, the phase contrast images that were simultaneously captured were slightly out-of-focus Localization of the Rod complex was then quantified by measuring the area on the image of fluorescence emitted from sfGFP-RodZ or sfGFP-RMR ( Figure A2). We found that the Rod complex containing RMR was significantly larger than that containing RodZ. These results suggest that the Rod complex containing RMR is somewhat different from that containing WT RodZ. Image analysis revealed that most RodZ or RMR colocalized with MreB, but the degree of colocalization was somewhat reduced for RMR compared with RodZ (Figure 2c,d; T A B L E 4 Growth rate, length, and width of cells carrying a suppressor mutation. | 9 of 23 Figure A3a,b). These results suggest that some RMR failed to form proper clusters, unlike WT RodZ, and may have led to cells producing RMR showing an abnormal shape and slow growth phenotype. It is possible that RMR lost abilities to interact with itself and other proteins so RMR failed to form proper clusters. Thus, we examined those interactions by the bacterial-two hybrid assay (BACTH assay) (Karimova et al., 1998). RMR retained abilities to interact with itself and other proteins although we could not detect the interaction between RodZ/RMR and PBP2 ( Figure A4a). This result suggests that RMRcano interacts with each protein but would not be able to organize the overall structure of the Rod complex. The transmembrane domain of RodZ likely plays a role in forming or stabilizing the proper structure of the Rod complex.
3.3 | Isolation of mutants suppressing the slow-growth phenotype of the rmr cells Previously, we isolated suppressor mutations of the slow-growth phenotype of ΔrodZ cells .
Most of the suppressor mutations were found in the components of the Rod complex. We expected that if we isolated suppressors of RMR cells, we would find mutations in the interaction sites between RodZ and other proteins, in addition to mutations in the components of the Rod complex. To isolate the suppressor mutants of RMR cells, several independent colonies of RU1353 (sfgfp-rmr) cells were grown in L medium at 37°C overnight. The cells were diluted in fresh L medium the next morning and grown the next day at 37°C, and this cultivation was repeated for 1 week. Then, the cells were plated on an L agar plate, and the plates were incubated at 37°C overnight. It is known that ΔrodZ cells which grow slower than WT cells form smaller colonies on the L agar plate (Shiomi et al., 2008. Thus, if larger colonies emerged, it would be suppressor mutants of the slowgrowth phenotype of RMR cells. Larger and smaller colonies emerged. We independently isolated 18 of these large-colony suppressor mutants. We could determine the mutation sites in 16 out of 18 suppressors by whole-genome sequencing. Some of the suppressor mutations isolated in this study had already been isolated in the previous study, in which we isolated suppressors of the slowgrowth phenotype of ΔrodZ mutant   (Table 5).
Unexpectedly, no mutations were found in the RMR itself. Instead, all of the mutations (15 mutations), except for one mutation, were found in mreB, mreC, mreD, mrdA encoding PBP2, or mrdB encoding RodA, which are involved in the Rod complex. We will report the suppressor mutation occurring outside the Rod complex in a separate paper.
Suppressor mutations were mapped onto a three-dimensional structural model of each protein (Figure 3). Many of the suppressor mutations were located at the protein-protein interaction surfaces, suggesting that the suppressors could have altered the protein-protein interactions of the Rod complex components to compensate for RMR. Indeed, we previously showed that MreB A125V exhibited stronger interactions with itself (self-interaction) and MreC than WT MreB . It was shown that the PBP2 L61R mutant suppresses a MreC defect (Rohs et al., 2018) and activates the GTase activity of RodA without changing the interaction of RodA and PBP2 (Liu et al., 2020). Therefore, it is plausible to hypothesize that PBP2 T52I and PBP2 I59S mutants activate the GTase activity of RodA because Thr52 and Ile59 in PBP2 are close to Leu61. We also examined interactions between PBP2 and RodA A234T or RodA K243N by the BACTH assay and found that RodA A234T and RodA K243N showed stronger interaction with PBP2 than WT RodA although we could not detect interaction between PBP2 and WT RodA ( Figure A4b). This would be consistent with the observation that RodA A234T has a stronger activity to synthesize peptidoglycan (Rohs et al., 2018). Therefore, these mutations may increase the activity of the Rod complex.
Before we analyzed the suppressor mutants, we transferred all of the suppressor mutations (mutations in mreB, mreC, mreD, mrdA, or mrdB) to WT and RMR strains. We also transferred the mutations into ΔrodZ cells to examine whether the mutations were capable of restoring the slow-growth phenotype of cells lacking rodZ. The growth rates were calculated (Table 4 and Figure A5). None of the suppressor mutations significantly affected the growth rate of the WT strain, but restored the slow-growth phenotype of RMR and ΔrodZ cells (Table 4 and Figure A5), indicating that the mutations isolated as suppressors of the slow-growth phenotype of RMR cells could also suppress the slow-growth phenotype of ΔrodZ cells.

| Characterizations of the suppressors
When we previously isolated the suppressors of ΔrodZ cells, the suppressor mutations restored not only the growth rate but also the cell shape. Therefore, we next observed the cell shape and measured the length and width of all strains carrying suppressor mutations ( Figure A6 and Table 4). We found that all the mutations completely or partially restored the rod shape, although the cell width of RMR and ΔrodZ cells was variable, with the distribution of the width of the suppressor strains producing RMR or lacking rodZ being relatively narrow.
To investigate whether these suppressor mutations restored Rod cluster formation, we observed the subcellular localization of RodZ and RMR (Figure 4 and Figure A2) and colocalization with MreB in cells producing suppressors ( Figure A3). As described above, sfGFP-RMR formed brighter and larger clusters. However, sfGFP-RMR in cells carrying suppressor mutations formed clusters similar to those of sfGFP-RodZ. We also quantitatively analyzed the Rod complex formation ( Figure A2) and colocalization between RodZ or RMR and MreB in the suppressors ( Figure A3). These results suggest that the suppressor mutations restored the assembly of the Rod complex even though the strains had RMR in the Rod complex.  . b These amino acids were mutated in suppressors of ΔrodZ, but the amino acids replaced were different . c mreB R124L mutation was isolated in two independent suppressors. We previously showed  that ΔrodZ cells are more sensitive to the antibiotics A22 (Iwai et al., 2002), which inhibit the binding of ATP to MreB (Bean et al., 2009) and mecillinam, which inhibits the transpeptidase activity of PBP2 (Spratt, 1975). We found that RMR cells were also more sensitive to both antibiotics ( Figure 5). If the suppressor mutations improved the assembly of the Rod complex, the cells would be resistant to these antibiotics. We examined the sensitivity to A22 of RodZ or RMR cells carrying suppressor mutations. As shown in Figure 5a or RodA K243N were more resistant to 1 or 5 µg/mL A22 than RodZ or RMR cells (Figure 5b), supporting the idea that these mutations increased the integrity of the Rod complex or the activity of the Rod complex. If these mutations increase the integrity of the Rod complex and therefore peptidoglycan synthesis activity is increased compared with that of RMR cells, the suppressor cells may also change the sensitivity to mecillinam, which specifically binds to PBP2. Thus, we examined the sensitivity of the suppressor cells to mecillinam ( Figure 5). RMR cells producing MreB E122D , MreB R124S , MreB A125V , MreB E137G , MreD F123L , all suppressor mutants in PBP2 except PBP2 A201V , RodA A234T , or RodA K243N were slightly more resistant to 0.1 µg/mL mecillinam than RMR cells. These suppressor cells may have increased peptidoglycan synthesis activity compared with that of RMR. It was shown that RodA A234T has an increased activity of the Rod complex (Rohs et al., 2018). However, RodZ cells producing MreB R124L were more sensitive to mecillinam than WT cells. RMR cells producing MreC S153I were more sensitive to mecillinam than RMR cells producing MreD F123L , suggesting that MreD F123L functions as a stronger suppressor of RMR than MreC S153I . The morphology of RMR cells producing MreD F123L is more similar to rod-shaped WT than that of RMR cells producing MreC S153I . showed a relatively homogeneous structure (Figure 6a,c, Figure A7, and Table 6). Many small holes were observed, as previously observed (Demchick & Koch, 1996; Pasquina-Lemonche et al., 2020). There was (a) (b) F I G U R E 5 Sensitivity of cells producing suppressor mutations to antibiotics. Sensitivity of cells producing mreB, mreC, or mreD (a) or mrdA (encoding PBP2) or mrdB (encoding RodA) (b) to A22 and mecillinam. An overnight culture of the indicated strains was diluted serially (from 10 −1 to 10 −6 ) and spotted onto L plates containing A22 or mecillinam. The plates were incubated for 24 h at 37°C. AGO ET AL.

| Peptidoglycan structure revealed by QFDE-EM
| 13 of 23 no significant difference in the pore size of the peptidoglycan of BW25113 (WT) (19.8 ± 28.6 nm 2 ) and RU383 (RodZ) (21.2 ± 31.7 nm 2 ) (p = 0.0066). The peptidoglycan purified from the RMR cells clearly had larger holes (42.2 ± 81.0 nm 2 ), and the number of holes was higher than that of the peptidoglycan purified from WT cells (Figure 6d, Figure A7 and Table 6), suggesting that the Rod complexes containing RMR synthesize aberrant peptidoglycan, leading to the abnormal shape. The pore size of the peptidoglycan of ΔrodZ (30.0 ± 51.0 nm 2 ) was smaller than that of RMR (Figure 6b,d and Figure A7) but larger than that of the wild strain. We observed the structure of peptidoglycan purified from RMR cells carrying a suppressor mutation.
The number of holes was clearly reduced compared with that of RMR peptidoglycan but was still higher than that in WT peptidoglycan (Figure 6e-k, Figure A7, and Table 6). The size of these holes except for RMR RodA A234T was almost the same as that in the WT peptidoglycan ( Figure A7 and Table 6). These results suggest that, in the suppressor strains, the activity of the Rod complex containing RMR was increased by strengthening the protein-protein interactions within the Rod complex, or by unknown mechanisms, thus allowing the synthesis of the correct peptidoglycan.

| Chemical structures of peptidoglycan
We showed by using QFDE-EM that the overall abnormal structures of the peptidoglycan from cells producing RMR reverted to the normal peptidoglycan structure with each suppressor mutation ( Figure 6). We next examined how the structures of muropeptides of RMR and suppressors differ chemically from that of WT by using LC/MS analysis of purified peptidoglycan. Here, we used BW25113 (WT) as a control strain and analyzed RU1666 (sfGFP-RMR MreB A125V ) as a suppressor Structures of peptidoglycan. (a-k) Peptidoglycan purified from the indicated strain was observed by quick freeze, deep-etch, and electron microscopy. Representative pictures are shown.
T A B L E 6 The number and size of holes in purified peptidoglycan. strain. Tri, Tetra, Tetra-Tetra, Tetra-Tri, and anhydro Tetra-Tetra muropeptides were the most abundant in RU1353 compared with other strains (Figure 7). Tetra-Tetra is a structure normally found in peptidoglycan. Therefore, it appears that the Rod complex is more active in RU1353 compared with RU383, contrary to our previous conclusion. On the other hand, other muropeptides are mainly present during and after cell wall repair, suggesting that peptidoglycan of RU1353 was damaged and repaired much compared with other strains.
It was shown that β-lactam antibiotics which inhibited the activity of PBPs induced a futile cycle of cell wall synthesis and degradation (Cho et al., 2014). Thus, the increase in Tetra-Tetra muropeptides in RU1353 was not simply an increase in the activity of the Rod complex but rather suggests that a futile cycle of cell wall synthesis and degradation was induced in RU1353. Furthermore, a comparison of the muropeptide composition of BW25113 and RU383 showed that each muropeptide was more abundant in RU383 (Figure 7). The difference between these strains is whether or not sfGFP is fused to the Nterminus of RodZ. The results, therefore, suggest that, although there are no major differences in the growth rate, morphology, or overall structure of the peptidoglycan between the two strains ( Figures A1   and A7), there are differences in the chemical structure of the peptidoglycan. In other words, fusing sfGFP to RodZ may reduce the activity of the Rod complex. Interestingly, the muropeptide compositions of RU1616 (sfGFP-RMR MreB A125V ) were rather closer to BW25113 (WT) than to RU383 (sfGFP-RodZ). Therefore, the MreB A125V mutation suppresses not only the reduced function of the Rod complex by RMR but also the reduced function of the Rod complex by the fusion of sfGFP.
An analysis of the function of each region of the transmembrane protein RodZ was performed previously (Bendezú et al., 2009;Shiomi et al., 2008). The N-terminal cytoplasmic region interacts with MreB, and the C-terminal periplasmic region interacts with RodZ, MreC, MreD, and PBP2 (Bendezú et al., 2009;van den Ent et al., 2010;Ikebe et al., 2018). In addition, the interaction between MreC and PBP2 may be important for the activation of the Rod complex Consistently, most of the suppressor mutations that restored the slow-growth phenotype and morphological abnormality of ΔrodZ were found in the components of the Rod complex . We constructed an RMR-producing strain to elucidate the function of the transmembrane region of RodZ and found that RMR cells are not as morphologically abnormal as ΔrodZ cells, but they are also not the same as WT cells. Furthermore, the growth of mutations were found. The absence of suppressor mutations in the cytoplasmic interaction site with MreB and the periplasmic interaction site with PBP2 in the RMR molecule suggests that a single mutation in one of these regions is not sufficient for suppression and that the transmembrane region of RodZ is important for the correct connection between the cytoplasmic and periplasmic regions in the Rod complex. Instead, most of the mutations were found in the Rod complex, such as mreB and mrdA (encoding PBP2). Some of these were already isolated as suppressors of ΔrodZ cells . When these mutation sites were mapped onto the structure of each protein, they were located at protein-protein interaction sites. MreB R124 and MreB A125V are located at the interface between the MreB filaments (van den Ent et al., 2014). It was shown that MreC is important for inducing conformational changes in PBP2, and growth defects caused by MreC G156D were suppressed by PBP2 T52A , PBP2 L61R , and RodA A234T (Rohs et al., 2018).
Furthermore, it was shown that PBP2 L61R and RodA A234T increased peptidoglycan synthesis activity (Rohs et al., 2018). We isolated mreC S153I , mrdA T52I , mrdA I59S , mrdB A234T , and mrdB K243N as suppressor mutations in RMR cells. These are located in the same regions as Peptidoglycan has been observed by EM and AFM, and its structures have been reported (de Pedro et al., 1997;Elsbroek et al., 2023;Gan et al., 2008;Pasquina-Lemonche et al., 2020;Salamaga et al., 2021;Tulum et al., 2019;Turner et al., 2018). In particular, QFDE-EM is an excellent tool for visualizing the structure of peptidoglycan with high resolution. Using this method, the cell surface of B. subtilis and the outer membrane vesicles of E. coli and their formation processes have been observed in detail (Ojima et al., 2021;Tahara & Miyata, 2023;Tulum et al., 2019). The relationship between the activity of the Rod complex and the overall structure of the peptidoglycan has been controversial. Turner et al. showed that the pore sizes of peptidoglycan are not different even after A22, an inhibitor of MreB (Iwai et al., 2002), was added to cells (Turner et al., 2018) while Elsbroek et al., showed that peptidoglycan became less dense when cells were treated with β-lactam antibiotics (Elsbroek et al., 2023). We isolated peptidoglycan from WT, RMR, and suppressor cells and visualized their structures at high resolution by QFDE-EM. Very small holes were observed in the peptidoglycan purified from WT. In our observation, the average size of the hole was~20 nm 2 . It has been reported that the radius of the hole of E. coli peptidoglycan is 2.06 nm (approximately 13 nm 2 in area) (Demchick & Koch, 1996). In other reports estimated by AFM, the diameter of the hole of E. coli peptidoglycan is 10 nm (approximately 79 nm 2 in area) (Elsbroek et al., 2023;Turner et al., 2013Turner et al., , 2018 Elsbroek et al. (2023) reported that the addition of β-lactam antibiotics made peptidoglycans less dense, that is, the pores became larger, in which the authors concluded that their results are consistent with the mechanism of action of β-lactam antibiotics to inhibit peptide cross-linking.
Although we do not know why these observations using AFM obtained different results, our results are consistent with those of Elsbroek et al. (2023) and are therefore consistent with our conclusion that the RMR-producing strain probably has reduced peptidoglycan synthesis activity. Furthermore, Salamaga et al. (2021) reported that peptidoglycan purified from S. aureus cells treated with methicillin or vancomycin had larger holes than those purified from nontreated cells, using AFM. Since methicillin and vancomycin inhibit peptidoglycan synthesis, it was concluded that the holes were generated because the hydrolytic activity of the peptidoglycan exceeded that of its synthesis. This result was in good agreement with our observations. We conclude that the Rod complex may be a determinant not only for the whole shape of peptidoglycan and cell morphology but also for its highly dense structure to support the mechanical strength of the cell wall.
Our LC/MS analysis of purified peptidoglycan revealed that Tri, Tetra, Tetra-Tetra, Tetra-Tri, and anhydro Tetra-Tetra muropeptides were the most abundant in RU1353 compared with other strains. The increase in Tetra-Tetra muropeptides, which are normally present in peptidoglycan, in RU1353 was not simply an increase in the activity of the Rod complex but rather suggests that a futile cycle of cell wall synthesis and degradation was induced in RU1353. The decrease in the activity of the Rod complex would lead to peptidoglycan damage and subsequent repair of peptidoglycan. This is consistent with the results of electron microscopic observation of peptidoglycan.
Furthermore, the suppressor mutation indeed suppressed the reduced activity of the Rod complex containing RMR. However, this analysis also yielded an unexpected result: although fusing sfGFP to RodZ did not significantly affect the growth rate, cell morphology or overall structure of the peptidoglycan in cells producing sfGFP-RodZ ( Figures A1 and A7), the compositions of the muropeptide were different (Figure 7). The results suggest that the activity of the Rod complex containing sfGFP-RodZ is slightly reduced compared with that of the wild-type strain. Observations of E. coli cells producing MreB fused with fluorescent proteins have been also reported (Bendezú et al., 2009;Ouzounov et al., 2016 coli has the robustness to retain its overall structure even if the peptidoglycan structure is somewhat changed.
In this work, we visualized the peptidoglycan of E. coli at high resolution without chemical treatment and analyzed the muropeptide compositions. In the future, by combining these methods, we would like to analyze mutant strains of factors involved in peptidoglycan synthesis, degradation, and repair to gain a macroscopic understanding of how each protein plays a role in maintaining the peptidoglycan structure. F I G U R E A6 Cell morphology of cells producing each suppressor. RMR, RodZ, or ΔrodZ cells producing each suppressor were grown to log-phase in L medium, and phase contrast images were taken.
F I G U R E A7 Size of holes in peptidoglycan. Violin plots showing the distribution of the size of holes in peptidoglycan purified from the indicated strains. Distribution of the size and number of holes in peptidoglycan purified from each strain. The size and number of holes in the images of the three peptidoglycans were quantified. Average and standard deviation (SD) are shown. p Values were determined by unpaired T test. ns: p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.